JP2000095800A - Antibody for activating erythropoietin receptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new antibody comprising an antibody (or its fragment) activating erythropoietin receptor, capable of activating erythropoietin receptor and stimulating erythrocyte formation, and useful as a therapeutic agent for anaemia, or the like. SOLUTION: This new antibody is an antibody (or its fragment) activating erythropoietin receptor (referred to as EPOR), capable of activating erythropoietin receptor and stimulating erythrocyte formation, and useful as therapy for anaemia, diagnosis of diseases due to dysfunctional EPOR, or the like. The new antibody is obtained as an antibody for stimulating the propagation of erythropoietin-dependent cells by testing an anti-EPOR monoclonal antibody prepared through immunization of soluble human EPOR against a mouse with respect to EPOR bonds according to realtime biospecific interaction analysis (BIAcore analysis) and by screening the bonds of EPOR to the surface of transfected CHO cells according to fluorescence activated cell sorter(FACS) analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an antibody that recognizes an erythropoietin receptor. Specifically, the present invention relates to antibodies that activate erythropoietin receptors and promote erythropoiesis.

[0002]

BACKGROUND OF THE INVENTION Erythropoietin (EPO) is a glycoprotein hormone involved in the proliferation of erythroid progenitor cells and the maturation of erythroid progenitor cells into erythrocytes. EPO is produced by the liver during fetal life and by the kidneys in adults, promoting the production of red blood cells from erythroid precursors. E commonly occurs in adults as a result of renal failure
Decreased production of PO causes anemia. EPO has been produced by genetic engineering techniques involving expression and secretion of this protein from host cells transfected with a gene encoding erythropoietin. Administration of recombinant EPO is effective in treating anemia. For example, Eschba
ch et al. (N. Engl J Med 316 , 73 (1
987)) describes the use of EPO to treat anemia due to chronic renal failure.

[0003] Purification of human urinary EPO has been described by Miyake et al. (J. Biol. Chem. 252 , 5558 (19).
77)). The identification, cloning and expression of the gene encoding erythropoietin is disclosed in US Pat. No. 4,703,008 (Lin). A description of a method for the purification of recombinant EPO from cell culture is given in US Pat. No. 4,667,016 (Lai et al.).
Included in.

Little is known about the mechanism by which EPO promotes erythropoiesis. It is clear that EPO binds to specific cell surface receptors to activate cells and promote their proliferation and / or differentiation, but the unique mechanism of this activation and the receptor And the structures of their respective related proteins are not completely understood. The erythropoietin receptor (EPO-R) is thought to exist as a multimeric complex. Sedimentation studies showed that the molecular weight was 330
± 48 kDa (Mayeux et al.,
Eur. J. Biochem. 194 , 271 (199
0)). From cross-linking studies, the receptor complex shows that at least two separate polypeptides, ie, 66-72
kDa and 85, 100 kDa species (Mayueux et al., J. Biol. Che).
m. 266 , 23380 (1991); McCaffe.
ry et al. Biol. Chem. 264 , 10507
(1990)). Another 95 kDa protein was also detected by immunoprecipitation of the EPO receptor (Miura and I).
hle, Blood 81, 1739 (199
3)). According to another cross-linking study, 110 kDa,
Three Es containing the 130 kDa and 145 kDa complexes
PO became clear. The 110 kDa and 145 kDa complex contained the EPO receptor because it was possible to immunoprecipitate it with an antibody against the EPO receptor (M
iura and Ihle, supra). Carboxy-terminal truncation E
The expression of the PO receptor detected a 110 kDa complex but not a 145 kDa complex. This suggests that the complex with a molecular weight of 145 kDa contains the polypeptide present in the 110 kDa complex and an additional 35 kDa protein.

[0005] Further insights into the structure and function of the EPO receptor complex have been obtained by cloning and expression of the mouse and human EPO receptors (D'A).
ndrea et al., Cell 57 , 277 (1989);
Jones et al., Blood 76 , 31 (1990); W.
inkelman et al., Blood 76, 24 (199
0); PCT Application No. WO 90/08822; D'An
U.S. Pat. No. 5,278,065 to drea et al.). The full-length human EPO receptor has 483 amino acids with an extracellular domain of about 224 amino acids and a signal peptide of 25 amino acids.
It is an amino acid transmembrane protein. The human EPO receptor shows about 82% amino acid sequence homology with the mouse receptor. Cloned full-length EPO expressed in mammalian cells
The receptor (66-72 kDa) has been shown to bind to EPO with an affinity (100-300 nM) similar to that for the native receptor on erythroid progenitor cells. Therefore, this configuration is the main EP
It is thought to contain the O-binding determinant and is called the EPO receptor. 85 kDa and 10 which are considered part of the crosslinked complex
The 0 kDa protein is distinct from the EPO receptor, but should be very close to EPO because it is able to crosslink EPO to these proteins. 8
The 5 kDa and 100 kDa proteins are related to each other, and the 85 kDa protein may be a proteolytic cleavage product of the 100 kDa species (Sawye
r, J. et al. Biol. Chem. 264 , 13343 (1
989)).

[0006] A soluble (truncated) form of the EPO receptor containing only the extracellular domain has been made, and this form of the receptor has an affinity of about 1 nM, ie, about 1 / l of the full length receptor.
It has been found to bind EPO with an affinity as low as 3 to about 1/10 (Harris et al., J. B.
iol. Chem. 267 , 15205 (1992);
Yang and Jones, Blood 82, 1713.
(1993)). The cause of the lower affinity than the full-length protein is unknown. Other protein species may be part of the EPOR complex and contribute to EPO binding, thereby increasing affinity. This possibility suggests that the fusion of a cell line with a low affinity EPO receptor with CHO cells that do not bind to EPO resulted in a hybrid cell line that exhibited a high EPO binding affinity of the receptor for EPO. And Goldwasher (E
xp. Hematol. 21 , 483 (1993)). In addition to this,
Transfection of OR into CHO cells resulted in a cell line with both high and low affinity receptors as quantified by Scatchard analysis. Amplification of EPOR copy number increased low affinity binding but did not increase high affinity binding. These results indicate that CH converts low affinity EPOR to high affinity.
This is consistent with the limited amount of protein present in O cells.

[0007] Activation of the EPO receptor produces several biological effects. Three of these activities are promotion of proliferation, promotion of differentiation, and inhibition of apoptosis (Li
Boi et al., Proc. Natl. Acad. Sci. U
SA 90 , 11351 (1993); Koury, S
science 248 , 378 (1990)). Signal transduction pathways that promote proliferation and differentiation are thought to be distinct from each other (Noguchi et al., Mo.
l. Cell. Biol. 8 , 2604 (1988);
Patel et al. Biol. Chem. 267 , 21
300 (1992); Liboi et al., Ibid.). Several results suggest that auxiliary proteins may be required to mediate the differentiation signal (Chiba et al., N.
atature 362 , 646 (1993); Chiba
Proc. Natl. Acad. Sci. USA
90 , 11593 (1993)). However, the role of accessory proteins in differentiation is controversial because the constitutively activated form of the receptor can promote both proliferation and differentiation (Pharr et al., Proc. N).
atl. Acad. Sci. USA 90 , 938 (1
993)).

[0008] Activation of the EPO receptor may be due to its dimerization. That is, EPO may act as a crosslinker between two EPO receptor molecules.
There is evidence to support this proposal. A constitutive activation of the receptor is obtained as a result of the arginine to cysteine mutation at position 129 of the mouse EPO receptor, presumably due to the disulfide bond formed between the two receptor subunits (Yoshimura Et Nat
ure 348 , 647 (1990)). In addition, EPOR has been found in multimeric complexes in cells (Miura and Ihle, Arch. Bioche).
m. Biophys. 306 , 200 (1993)). However, the isolation of stable multimeric forms of the purified EPO soluble receptor has not been reported to date. In addition to this, EP
Although dimerization of the OR is required, this dimerization alone may not be sufficient for full cell activation. For example, dimerization produces a proliferation signal, but not a differentiation signal. That is, an auxiliary protein may be required in order to send a differentiation signal.

[0009] The relationship between dimerization and activation of the EPO receptor can be used to identify compounds that differ from EPO but activate the EPO receptor. For example, an antibody has two identical binding sites for an antigen. Anti-EPOR antibodies are capable of binding two EPOR molecules, bringing these molecules into close proximity with one another, resulting in dimerization. To function in vivo, these antibodies must recognize EPOR on the cell surface and bind to allow activation of the signal transduction pathway. In addition, it is desirable that both proliferation and differentiation of erythroid progenitor cells occur as a result of activation. Human growth hormone receptor (Fuh et al., Sc
gene 256 , 1677 (1992)) and epidermal growth factor receptor (Schreiber et al., Proc. Na.
tl. Acad. Sci. USA 78 , 7535 (19
A similar approach for understanding the activation of 81)) has been reported.

[0010] It would be desirable to identify molecules with properties that activate the EPO receptor and stimulate erythropoiesis. In order to make this identification, it is important to understand the mechanism of EPO receptor activation and signal transduction. One approach to elucidating this mechanism would be to identify antibodies that recognize the EPO receptor so as to activate the EPO receptor and stimulate erythropoiesis. Such antibodies will be useful for therapeutic and diagnostic applications, and will also be useful for examining EPO receptor function.

The following several references describe antibodies that bind to the mouse or human EPO receptor.

[0012] D'Andrea et al. (The Biolo)
gy of Hemtaopoiesis, Wiley
-Liss, Inc. (1990) pp. 153-1
59) produced a polyclonal anti-peptide antibody against the amino and carboxy terminal peptides of the mouse EPO receptor. Western Blot demonstrated that this antibody reacted with the mouse EPO receptor.

[0013] Bailey et al. (Exp. Hemato
l. 21 , 1535-1543 (1993)) have produced polyclonal anti-peptide antibodies against a synthetic peptide having homology to the extracellular and cytoplasmic domains of the mouse EPO receptor. Receptor activation by these antibodies was not detected as measured by 3H thymidine incorporation into spleen cells from phenylhydrazine-treated mice.

(Baynes et al., Blood 82 , 20)
88-2095 (1993)) have produced polyclonal antibodies to the amino-terminal peptide in the human EPO receptor. This antibody was shown to react with a soluble form of the EPO receptor present in human serum. D '
Andrea et al. (Blood 82 , 46-52 (19
93)) produced a monoclonal antibody against the human EPO receptor. These antibodies are human EPO cDN
Binds to Ba / F3 cells transfected with clone A, some of which antibodies inhibit EPO binding and
Dependent growth was neutralized.

(Fisher et al., Blood 82 , 19).
7A (1993)), to distinguish erythroid progenitors that undergo EPO-dependent growth and maturation from erythroid progenitors that undergo EPO-independent growth and maturation, D'And
The same monoclonal antibodies as those described in the above reference to Rea were used.

[0016]

Activating the EPO receptor or stimulating the proliferation and / or maturation of erythroid progenitor cells has been reported for any of the antibodies described in the above references. Not.

Accordingly, it is an object of the present invention to produce an antibody that recognizes and binds to an EPO receptor such that the EPO receptor is activated. Yet another object of the present invention is to produce an antibody that promotes erythropoiesis by binding to the EPO receptor and stimulating erythroid progenitor cell proliferation and / or differentiation into erythrocytes. This antibody can be used to treat anemia, or dysfunctional EPO
It is effective in diagnosing a disease characterized by a receptor. In addition, this antibody will allow the identification of therapeutic agents for the treatment of anemia.

[0018]

SUMMARY OF THE INVENTION The present invention relates to an antibody or a fragment thereof that activates an erythropoietin receptor. By screening for antibodies recognizing the human EPO receptor, two antibodies named "Mab71" and "Mab73" were grown on UT7-EPO cells (an EPO-dependent cell line that would not grow in the absence of added EPO). It turned out to be irritating. In addition, Mab 71
Erythroid colony formation from erythroid progenitor cells in human blood was stimulated. Antibodies within the scope of the present invention are Mab
71 or Mab 73 may recognize an epitope on the EPO receptor. Preferably, the antibodies of the present invention are monoclonal antibodies, which can be humanized or human. Furthermore, included within the scope of the invention are hybridoma cell lines that produce the antibodies of the invention.

The present invention further provides a method and a kit for detecting an EPO receptor in a biological sample, the kit comprising the EPO receptor antibody of the present invention. Pharmaceutical compositions comprising an EPO receptor antibody of the present invention and a pharmaceutically acceptable adjuvant are also included within the scope of the present invention. Such compositions can be used to treat patients having a disease characterized by low red blood cell levels.

[0020]

DETAILED DESCRIPTION OF THE INVENTION A monoclonal antibody (Mab) that recognizes the erythropoietin receptor was produced by immunizing mice with purified soluble human EPO receptor. The soluble human EPO receptor was expressed and purified as described in Examples 1 and 2. Enzyme antibody method (ELI
Ma reacted with soluble human EPO receptor in SA)
b, 96 M for further screening
ab was selected. These Mabs were tested for EPO receptor binding by BIAcore analysis (Example 4).
A) And binding to the EPO receptor on the surface of transfected CHO cells was tested by FACS (Example 4C). Table 1 shows the results of this screening. Some of the antibodies bound to the EPO receptor were BIAc
As determined by ore analysis, only 5 of the 96 tested tested bound to the EPO receptor appearing on the surface of the transfected CHO cells as determined by FACS scanning. Twenty-four antibodies that were positive in the ELISA assay (including five antibodies that were positive in the FACS scan) were tested for stimulation of UT7-EPO cell proliferation. Surprisingly, "Mab
Two antibodies named "71" and "Mab 73"
The UT7-EPO cell line (Ko) in the absence of EPO
Matsu et al., Blood 82 , 456 (199).
3)) stimulated the incorporation of ³ H-thymidine into it (Example 8A). The growth of this UT7-EPO cell line requires the presence of EPO in the medium. Therefore, the stimulation of UT7-EPO cell proliferation is likely to be due to EPO receptor activation by Mab 71 and Mab 73. As shown in FIG. 2, the response of UT7-EPO cells was higher when Mab71 was present than when Mab71 was present.
3 was greater than if present. In addition, Mab 7
1 was found to promote erythroid colony formation from human erythroid precursors (see Example 9). This is the first case of an antibody that stimulates erythroid colony formation from human erythroid precursors.

The present invention provides an antibody or a fragment thereof that activates an erythropoietin receptor. As used herein, the term "activation of an EPO receptor" refers to an EPO that causes a signal to be introduced inside a cell bearing the receptor.
Refers to one or more molecular processes performed by a receptor, in which a signal ultimately results in one or more cell physiological changes. The cellular response to EPO receptor activation is typically a change in the growth or differentiation of cells bearing the receptor. Cells having the receptor are generally erythroid progenitor cells.
lls). At this time, the molecular events that cause signal transduction by the EPO receptor are poorly understood. However, as described in the background of the invention, EP
Some evidence indicates that O receptor dimerization is at least one event that may be required for activation. The disclosure of the present invention also provides support for this idea. As shown in FIG. 5, replacement of Mab 71 with the corresponding Fab fragment called Fab 71 abolishes Mab 71 stimulation of ³ H-thymidine incorporation into UT7-EPO cells.
Thus, a complete bivalent antibody with the corresponding monovalent fragment eliminates the proliferative response. In addition to this, Mab
71 inhibits EPO receptor activation at high concentrations. Both of these observations support a dimerization model of EPO receptor activation. Mab 71 is human EPO-
It was shown to interact with the synthetic peptide at residues 49-78 of R (see Example 6). Therefore, this EP
The OR region can result in activation of EPO-R when joined by a crosslinker such as Mab 71. Molecules that bridge two EPO-R molecules by binding to residues 49-78 also
It should be understood that they fall within the scope of the present invention.
Such molecules bridge the two EPO receptors by binding to residues contained within the region between residue 49 and residue 78, thereby causing dimerization and activation of the EPO receptor Or other bivalent molecules having the following characteristics:

The EPO receptor of the present invention is preferably a mammalian EPO receptor, and in a particularly preferred embodiment, a human EPO receptor. It should be understood that analogs of the human EPO receptor are also included within the scope of the present invention. Such analogs include amino acid insertions, deletions, extensions (exte) in the human EPO receptor sequence.
nsion) or by substitution. EPO
Examples of -R analogs are disclosed in U.S. Patent No. 5,292,654 (Yoshimura et al.), In which the substitution of a cysteine residue at position 129 of the EPO-R amino acid sequence is constitutively described. Activated EPO-R
Bring. Generally, human EPO having amino acid changes in regions other than the antibody binding domain required for activation
EPO-R analogs that retain the secondary and tertiary structure of the receptor can be recognized by the antibodies of the present invention. Mab 71 was shown to interact with a synthetic peptide at residues 49-78 of human EPO-R (see Example 6). Thus, an EPO-R analog that has changes in amino acid residues other than amino acid residues 49 to 78 and retains the secondary and tertiary structure of the EPO receptor is an MPO
ab 71. As used herein, numbering of amino acid residues in the human EPO-R polypeptide begins with proline at position 1, the amino terminal residue after cleavage of the 25 amino acid signal peptide.

The antibodies of the present invention bind to an epitope of the EPO receptor involved in receptor activation. In one embodiment, the antibody is EPO recognized by Mab 71
Recognizes an epitope on the receptor or an epitope recognized by Mab 73. Mab 71 converts amino acid residues 49 to 78 in human EPO-R.
Recognize a range of synthetic peptides. Therefore, Mab 71
May recognize an epitope on EPO-R defined entirely or partially by this sequence. The term "epitope" as used herein refers to a region of EPO-R where an antibody binds to this region and this binding prevents the binding of a second antibody to EPO-R.

The present invention also provides polyclonal antibodies, monoclonal antibodies, and fragments thereof.
Antibody fragments include those that activate the EPO receptor. Also includes humanized antibodies, typically produced by recombinant methods, in which the human sequence is
Includes part or all of an antibody that activates the EPO receptor. Examples of humanized antibodies include chimeric or CDR-grafted antibodies (US Pat. Nos. 4,816,567 and 5,225,539). Also included in the antibodies of the invention are fully human antibodies against the EPO receptor produced in genetically engineered mice (PCT Application No. 93/1).
No. 2227). The antibodies of the present invention can also have a detectable label attached to the antibody. Such labels include, for example, fluorescent labels (eg, fluorescein isothiocyanate, FITC), enzyme labels (eg, horseradish peroxidase), affinity labels (eg,
Biotin) or isotope labeling (eg, ¹²⁵ I).

Hybridoma cell lines that produce monoclonal antibodies that activate the EPO receptor are also included in the present invention. In one embodiment, the hybridoma cell line comprises:
EP recognized by Mab 71 or Mab 73
It produces a monoclonal antibody that recognizes an epitope on the O receptor. Generation of a hybridoma cell line producing a monoclonal antibody against human EPO-R is described in Example 3
Will be described. The hybridoma cell line that produces Mab71 is an American-based hybridoma cell line as of July 26, 1994.
Type Culture Collection,
Rockville, MD, under the accession number HB11689. The hybridoma cell line producing Mab 73 has been identified as Ame on July 26, 1994.
rican Type Culture Collec
accession number HB1 to Tion, Rockville, MD
Deposited as 1690.

The antibodies of the present invention are useful in diagnosing anemia and other diseases characterized by dysfunctional EPO-R. In one embodiment, a method of detecting an EPO receptor that can be activated in a biological sample comprises:
(A) contacting the sample with an antibody that activates the EPO receptor; and (b) detecting activation of the EPO receptor by the antibody. Biological samples include tissue specimens, whole cells, or extracts thereof. The antibodies of the invention can also be used as part of a diagnostic kit for detecting the presence of an EPO receptor in a biological sample. This kit uses an antibody conjugated with a label that allows detection.
The antibodies of the present invention are useful for identifying normal or abnormal receptors. The presence of the aberrant receptor in the biological sample
It may be an indicator of a disease such as Diamond-Blackfan anemia that is considered to be PO receptor dysfunction.

The antibodies of the present invention are useful in treating diseases characterized by low red blood cell levels. EPO in mammals
Method for modulating the endogenous activity of a receptor, preferably EPO
Methods for increasing the activity of the receptor are included in the present invention.
Generally, any condition treatable by erythropoietin (eg, anemia) is treatable by the antibodies of the present invention. The therapeutic antibody is administered in a dose and a route suitable for the type and severity of the disease to be treated, and such a dose and route can be appropriately determined by those skilled in the art. Administration by subcutaneous, intramuscular or intravenous injection is preferred.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody that activates EPO-R together with a pharmaceutically acceptable adjuvant, wherein the adjuvant comprises one or more diluents, carriers, preservatives. , Emulsifiers, antioxidants, and / or stabilizers. The term “therapeutically effective amount” as used herein refers to an amount of an antibody that produces a therapeutic effect for a given disease and dosing regimen. In the present invention, the therapeutic effect is a stimulation of erythropoiesis as evidenced by an increase in hematocrit in the patient to be treated. In a preferred embodiment, the antibodies of the invention are humanized or human antibodies that can be prepared using methods known to those skilled in the art. Pharmaceutically acceptable adjuvants are known to those skilled in the art,
n's PharmaceuticalScience
s, 18th Edition, A. R. Gennaro, Mack, E
Aston, PA (1990).

The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the invention.

[0030]

【Example】Example 1 Production of soluble human erythropoietin receptor A. For expression of soluble human erythropoietin receptor
Clone isolation  Humans as described by Jones et al. (Supra).
Using a clone containing the toerythropoietin receptor,
By CR method, soluble human erythropoietin receptor
A clone for expression of (sHuEPOR) was obtained. Hi
Primer for PCR amplification of toerythropoietin receptor
Marker 5 'primer: CTC CAA GCT TGC CGT CAC CAT GGA CC
A CCT CGG GGC GTC CCT (SEQ ID NO: 1) and 3 'primer: CAG GTC TAG ATT ACT AGG GAT CCA GG
T CGC TAG GC (SEQ ID NO: 2).

Plasmid 2.5 containing human EPO-R
ng, each of the above oligonucleotide primers 5 pm
ol, Tris-HCl (pH 8.3) 10 mM, KCl
50 mM, MgCl ₂ 1.5 mM, each dNTP 20
A PCR reaction was generated using 0 μM and 1 unit of Taq polymerase. 5 cycles of “94 ° C. for 30 seconds, 50 ° C. for 1 minute, 72 ° C. for 1 minute”, followed by 20 cycles of “94 ° C. for 30 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute” Amplification was performed by
The DNA was applied to a G-50 size exclusion column (Boehring).
er Mannheim Corp. ) And purified by digestion with Hind III and XbaI,
The expression vector pDSRa2 (DeClerck et al., J. Am.
Biol. Chem. 266, 3893 (1991))
Was connected. A clone containing the desired insert fragment is
Confirmed by sequence analysis. The d40EPOR clone was generated by PCR from a full length human EPOR clone (see above). The carboxy terminus of d40EPOR is tyr467, which is the result of the addition of a stop codon in the primer. Primers for PCR amplification are as follows: 5 'primer: 5'-CTC CAA GCT TGC CGT CAC CAT GGA
CCA CCT CGG GGC GTC CCT-3 '(SEQ ID NO: 1) and 3' primer: 5'-AGG TCG ACT ACT AGT AGT CAG TTG
AGA-3 ′ (SEQ ID NO: 3).

The PCR amplification was performed using pfu buffer 2 (Strata
tage, La Jolla, Calif.)
Polymerase was used. The reaction conditions were one cycle of “96 ° C. for 30 seconds, 45 ° C. for 1 minute, 72 ° C. for 1 minute”, followed by 25 cycles of “96 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. For 2 minutes. " Thereafter, a final 72 ° C. incubation was performed for 5 minutes. Reaction products were separated by agarose gel electrophoresis, and a band of about 1.3 Kb was isolated using a gene clean kit (BIO 101, Vista, CA). The purified fragment was subjected to PCR II (TAclo
ning kit, Invitrogen, SanDi
ego, CA). Recombinants were identified by restriction analysis and sequenced to confirm that the desired insert was present. The Hind III-SalI fragment was isolated as described above and ligated into the isolated pDSRa2 vector which had been previously cut with Hind III and SalI.
The resulting vector pDSRαEPORd40 was
Used for expression in O cells.

[0033]B. Soluble human EPO in CHO cells
Expression of R and d40 EPOR  The expression plasmid pDSRα2-EPOR-X was purchased from Jon.
As described in es et al. (supra), human EPOR
No. 1 contains the sequence encoding the acid Met1-Pro249.
Plasmid pDSRaEPORd40 contains Met1-T
Includes a sequence encoding yr467. Each plasmid 1
0 microgram of calcium phosphate mediated transfer
And introduced separately into CHO cells (W
Igler et al., Cell11, 233 (197)
7)). Individual colonies were excised from the vector
Selection was based on the expression of the lofoate reductase gene.
Expression of human EPOR by RNA hybridization
(Hunt et al., Exp. Hematol, 19: 779.
(1991)) and affinity purified antibodies were used.
Detection was performed by Western immunoblotting.
Cell lines that are positive in these assays can be expanded further
Selected for. For promoting the amplification of EPO-R expression
For this, the cell line was converted to 30 nM methotrexate (Mtx)
Adapted.

The preparation of the conditioned medium containing the soluble human EPOR was performed using a roller bottle and a hollow fiber bioreactor.
r). Roller bottle with growth medium (D
MEM: Non-essential amino acids (NEAA) and 30 nM Mt
x with 5% fetal bovine serum (FBS)
12 (1: 1) medium (GIBCO, Grand Isl)
and reagents from NY)) 2 × 10 ⁷ cells in 200 mL were inoculated. When confluence was achieved in 3-4 days, DMEM (Ham's F12 medium, NEAA, 30n
MMtx, serum-free). 6-7
After one day, the conditioned medium was collected and replaced with fresh serum-free medium. The second and third harvests were collected.

Cell Pharm bioreactor cartridges were inoculated with 5 × 10 ² cells in growth medium (as described above) supplemented with 5 μg / mL gentamicin. The pH was maintained at 7.3. Cells were depleted of serum starting 12 days after inoculation to prepare serum-free conditioned media. Collection of the conditioned media began on day 17.

[0036]Example 2 Purification of soluble human erythropoietin receptor  Make four different preparations of soluble recombinant human EPOR
Made. In the first preparation, the epoxy-activated Sephar
oose 6B (Pharmacia, Piscataw
ay, NJ) according to the manufacturer's instructions.
Bound with erythropoietin (rHuEPO). 32
mM ZnCl₂ RHuEPO 21 in 4.5 mL
8 mg prehydrated and H₂Epoxy active washed with O
Add to 7.2 g of sex Sepharose 6B. This
Of the slurry to a pH of 10.8 and then at room temperature
Mix overnight. Afterwards, ethanol to a final concentration of 1M
By adding the amine, any remaining reactive groups are blocked.
Lock and mix for 4 hours at room temperature. 8 ° C ±
Performed at 2 ° C. Combined resin (epoxy-EPO)
Fill into a ram and add 0.5M NaCl / 0.1M HO
Ac (pH 4) and 0.5M NaCl / 0.1M
Wash with an alternate cycle of borate (pH 8). Kara
The solution was adjusted to 140 mM NaCl / 10 mM Tris pH
Equilibrate at 7.6 (TBS). This column contains soluble
CHO cell expressing EPO-R (sHuEPO-R)
1560 m conditioned medium prepared with roller bottles from vesicles
Load L. After loading is completed, the column is
M NaCl / 10 mM Tris pH 7.6 (TBS)
And then bind sHuEPOR with 1M Na
Dissolved in Cl / 3M urea / 10mM Tris pH 7.6
Put out. Two UV_{28 0}Absorption peaks appear in this buffer
Therefore, it elutes. eluted second containing sHuEPOR
Pool of peaks and H₂Dilute 20-fold with O. Dilution
Pool from Mono Q (Pharmacia)
Load 1 mL pre-packed column and add 10 mM Tris pH
Elute with a NaCl gradient in 7.6. Single peak dissolution
Effluents were pooled, aliquoted and stored frozen at -80 ° C.

In the second preparation, a larger epoxy-E
Make a PO column. Epoxy activated Sepharo
Hydrate 20.4 g of se 6B, wash with H ₂ O, then with acetone, and finally with 50% formamide in H ₂ O (pH 10.6). H ₂ O 15
729 mg of rHuEPO in mL are titrated to pH 10.6, added to the resin and mixed overnight at room temperature. Thereafter, any remaining reactive groups were blocked by adding ethanolamine to a final concentration of 1M and at room temperature for 14 hours.
Mix for 0 minutes. Subsequent steps are performed at 8 ° C ± 2 ° C. Fill the column with epoxy-EPO and add 3M urea / 750
Wash with mM NaCl / 10 mM Tris pH 7.6, then equilibrate the column with TBS. sHuE
100 mL of conditioned medium prepared in a bioreactor from CHO cells expressing POR was added to Q Sepharo.
se Fast Flow (Pharmacia) 2m
Mix with L. The mixture is mixed at 8 ° C with frequent mixing.
Incubate at ± 2 ° C. for 30 minutes, then 0.45
Micron cellulose nitrate bottle top filter (Co
(ringing). Furnace liquid is epoxy-EP
Load on O column, 250 mM NaCl / 10 mM
Wash with Tris pH 7.6, then 3M urea / 750 mM NaCl / 10 mM Tris pH 7.
Elute at 6. The elution peak are pooled and diluted 20 fold with _{H 2} O. The diluted pool is added to Q Sepharose
Pack into a Fast Flow 15 mL column and add 10
Elute with a gradient of NaCl in mM Tris pH 7.6. The single eluted peak was pooled, aliquoted and
Stored frozen at ℃.

The third preparation uses the same epoxy-EPO column used in the second preparation. sEPO-
Roller bottle preparation from R-expressing CHO cells 850 mL of conditioned medium was added to Q Sepharose Fas.
Mix with 1.7 mL of tFlow. This mixture is processed in the same manner as in the second preparation.

In the fourth preparation, 7.25 L of a conditioned medium for preparing a bioreactor from CHO cells expressing sHuEPOR was added to Q Sepharose Fast F
Mix with low 110 mL. The mixture is incubated at 8 ° C. ± 2 ° C. for 1 hour with frequent mixing before being filtered through a 0.45 micron cellulose nitrate bottle top filter.

The filtrate was diluted with 7.25 L of H ₂ O,
Q Sepha equilibrated with mM Tris pH 7.6
Load into a 770 mL column of rose Fast Flow. Elute from the column with a gradient of NaCl in 20 mM Tris pH 7.6. Fractions containing large amounts of sHuEPOR based on SDS-PAGE analysis are pooled. Solid (NH ₄ ) ₂ SO ₄ is added to the pool to a final concentration of 1.2 M and then filtered through a 0.45 micron cellulose nitrate bottle top filter. The filtrate was washed with Phenyl Sepharose 6 (low s
ub, Pharmacia), 1.2 M → 0 M in 20 mM Tris pH 7.6.
Elute with a (NH ₄ ) ₂ SO ₄ decreasing gradient. The major eluting peak was _pooled, and the _{(NH 4)} in 2 SO ₄ 2.4M,
Precipitate sHuEPORt. The precipitated sHuEPOR is collected by centrifugation, resuspended in H ₂ O and titrated with Tris-HCl to pH 7.9. The resulting solution was added to 0.45
Filtered through a micron cellulose nitrate filter, aliquoted and stored frozen at -80 ° C.

[0041]Example 3 Preparation and screening of hybridoma cell lines A. Enzyme antibody assay (EIA)  First, the titer of serum antibody (Ab) of each animal is determined.
And then screen the available hybridomas.
EIA was performed to perform the printing. Flat bottom, high coupling, 96 holes
Microtitration EIA / RIA plates (Costar
Corporation, Cambridge, MA)
With a carbonate-bicarbonate buffer (pH 9.2) (0.0
15M Na₂CO₃, 0.035M NaHCO₃)
Coating with 5 μg of purified sHuEPO / mL
I did it. 50 μL of the Ab was added to each well. after
Plates are made of acetate film (ICN Biomed)
icals, Inc. , Costa Mesa, CA)
Cover with a rocking platform
rm) for 2 hours at room temperature (RT) or 1 hour at 4 ° C.
Incubated overnight. sHuEPOR lot # 1
Used after the first and second boosts and lot # 2
Used after 3 boosts. sHuEPOR lot
# 3 and Lot # 4 for screening hybridomas
Used for. BSA dilution / blocking solution concentrate
(Kirkegaard and Perry Lab
oratories, Inc. 1) Deionized water (d
H₂O) 5% BS prepared by mixing with 1 part
A solution using 250 μL per well, 30 RT at RT
Blocked for minutes. I removed the blocking solution
And a two-fold dilution of serum (1: 400 to 1: 5120)
0) Alternatively, the hybridoma tissue culture supernatant was
Added to the hole. Serum diluent is 1% BSA (Dulbec)
co-phosphate buffered saline (D-PBS) (Gibco B
RL, Grand Island, NY).
10% BSA dilution / blocking concentrate)
On the other hand, the hybridoma supernatant was tested undiluted.
Tested. In the case of a hybridoma test, one hole is connected.
As a conjugate control, another well was positive A
b served as control. RT while holding the plate again
For 1 hour, followed by dH₂Washing in O
Purified solution 20x concentrated solution (Kirkegaardand Pe
ry Laboratories, Inc. ) 1x
The preparation was washed four times. Then 1% BSA
Goat anti-mouse IgG heavy chain diluted 1: 1000 with
And light chain specific horseradish peroxidase-conjugated second Ab
(Boehringer Mannheim Bioc
chemicals, Indianapolis, IN)
Was incubated for 30 minutes in each well. As above
Wash the plate and dry by blotting
ABTS peroxidase single component substrate (Kirk
egard and Perry Laborato
ries, Inc. ) Was added. Microplate
 EL310 reader (Bio-tek Inst)
comments, Inc., Inc. 4) for each hole using
The absorbance was read at 05 nm. "Serum dilution" vs. "4
Log of "Optical Density at 05nm"₁₀And plot
50% point of the maximum optical density obtained by serum
Calculate half-maximal titers of serum antibodies by extrapolating with
did. Optical density is greater than 5 times background
In some cases, hybridomas were selected as positive.

[0042]B. Immunization  4.5 week old Balb / c mice (Charles R
vers Laboratories, Wilmin
gton, MA) for 10 animals
Duvant (CFA; 50% v / v; Difco Lab
oratories, Detroit, MI)
Clouded sHuEPOR (Lot # 1) (antigen) 50μ
g was injected subcutaneously (SQI). Four weeks later, Freun
d Incomplete adjuvant (ICFA; Difco Lab
oratories, Detroit, MI)
(Ag; lot # 1) 2 prepared as above
The animals were boosted with 5 μg (SQI). 9th
Later, mouse blood was collected from the tail and serum antibody (Ab)
The titer was quantified by an enzyme antibody assay (EIA). each
The half-maximal titer of each mouse increases to about 5000,
Animals were selected for hybridoma preparation. Ha of purpose
Used to make hybrids (# 71A and 73A)
For the three animals used (# 7, # 8, # 9), 1
2.5 μg Ag (lot 1) and 25 μg Ag (lot
2) and separately used at week 5 and week 29
It was necessary to do a boost. These boosts
Is similar to the initial boost, ie, 50% v / v I
Performed using an emulsion in CFA. Serum Ab titer
Monitoring continued 9 days after each boost. These
The final titer before fusion of the mouse was 5026 for animal # 7, dynamic
For object # 8, 6842, and for animal # 9, 12,945.
Was.

[0043]C. Cell fusion  Eight weeks after the final boost, sHuEPOR (lot #
3) Inject 25 μg intravenously into animals # 7, # 8, # 9
Was. Four days later, the mice were killed with carbon dioxide and
00U / mL penicillin G and 200μg / mL sulfuric acid
Du containing streptomycin and 4 mM glutamine
Ibecco's modified Eagles medium (2x P / S /
G DMEM) Take the spleen under sterile conditions into 25 mL.
Was. Remove excess fat tissue from the spleen and remove
Three discs containing “2x P / S / G DMEM”
And then washed through. Next, "2x P / S / G
 Sterile stomacher bag containing 10 mL of "DMEM"
(Stomacher bag) (Tekmar, Ci
ncinnati, OH) and transferred to Stomac.
her Lab Blender 80 (Secard
 LaboratoryUAC House, London
on, England) and single cell suspension
Liquid. When cells are released from the spleen wrap into the medium
Remove the cells from the bag and use 70 μm nylon
Mesh cell strainer (nylonmesh ce)
II strainer) (Becton Dicki
nson and Company; Lincoln
Park, NJ). The medium in the bag
Replace with fresh medium and release all cell contents of the spleen.
The above procedure was continued until it was completed. These spleen cells
Wash three times by centrifugation at 225 × g for 10 minutes.
Was. The first fusion used spleen cells from animal # 9
Was. In the second fusion, spleen cells from animal # 7 and animal # 8
The spores were pooled.

At the same time, complete medium (DMEM, 10
% Fetal bovine serum, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10 mM
Hepes buffer; Gibco Laborat
ories, Inc. , Grand Island, N
Y) Sp2 / 0-Ag14 mouse myeloma cells grown in (American Type Culture)
(Available from Collection, Rockville, Md. Under accession number CRL 1581) was washed as above. From this myeloma cell population, 4 × 10 ⁷ cells (fusion 1) or 8 × 10 ⁷
Individual cells (fusion 2) were harvested, mixed with the spleen cell suspension and pelleted again. The medium is aspirated from the cell pellet and polyethylene glycol (PEG 1500 MWt; Boehri) at 37 ° C. for “Fusion 1”.
nger Mannheim Biochemical
s, Indianapolis, Ind.) and for "Fusion 2," 3.5 mL of the same PEG were gently mixed into the medium over 1 minute. Thereafter, an equal volume of "2x P / S / G DMEM" was slowly added. The cells are allowed to stand at 37 ° C. for 2 minutes, after which additional “2”
xP / S / G DMEM "9 mL was added. The cells were again placed at 37 ° C. for 4 minutes.

Finally, "2x P / S / G DMEM"
30 mL was added to the cell suspension and cells were pelleted by centrifugation. Aspirate medium from pellet and remove cells,
It was gently resuspended in about 56 mL (fusion 1) or about 74 mL (fusion 2) of complete medium containing 100 U / mL penicillin G and 100 μg / mL streptomycin sulfate. Ten 96-well flat-bottom tissue culture plates (Becton) were added drop by drop from a 5 mL pipette.
Dickinson Labware; Lincoln
Park, NJ). Plate 37
Incubation was carried out in a humidified environment overnight at 5 ° C. and 5% CO ₂ . The next day, an equal volume of selective medium was added to each well. The selection medium is 0.1% in complete medium.
It consisted of mM hypoxanthine, 4 × 10 ⁻⁴ mM aminopterin and 1.6 × 10 ⁻² mM thymidine. The fusion plates were incubated for 7-10 days, and the medium was changed twice during this incubation period.
HAT selection medium was used after each fluid exchange. Tissue culture supernatants were collected from individual wells containing the hybrids and specific antibody activity against sHu EPOR was determined by EIA.
Inspected. Further screening was performed on 96 wells that were positive in EIA.

[0046]D. Dot blot  Dot blot of reduced sHuEPOR (Lot # 4)
For secondary screening of EIA-positive hybridomas
Used as method. Dot Blot SF Micr
Iteration Apparatus (Bio-
Rad Laboratories, Inc. , Ric
hmond, CA) with its instruction manual
Prepare according to the following procedure and prepare a nitrocellulose membrane (9 × 12 c
m, Bio-Rad Laboratories, In
c. , Richmond, CA) was used. First
Next, Tris-buffered saline (TBS; 10 mM Tris pH
7.5, 154 mM NaCl, 0.01% w / v
2-mercaptoethanol (5% v
/ V, Bio-Rad Laboratories, I
nc. , Richmond, CA) under reducing conditions.
The antigen was prepared by boiling for 5 minutes. sH
Load 25ng of uEPOR (Lot # 4) into each hole
And aspirate through the nitrocellulose membrane for binding
Was. 250 μL Blotto-Tween solution (broth)
Solution: 2% w / v skim milk powder, 50 mM Tris pH
7.5, 25 mM NaCl, 0.1 mM EDTA,
0.09% v / v Tween 20, 0.01% v / v
v Load antifoam A) into the hole and incubate at RT for 30 minutes.
I was vate. Aspirate the block liquid from the hole and repeat this procedure for 2
The non-specific sites on the nitrocellulose membrane
Completely blocked. After that, 0.1% v / v
Lioxyethylene sorbitan monolaurate (Twee
n-20; Bio-Rad Laboratorie
s, Inc. , Richmond, CA).
Washed three times through the membrane using BS. the next one
Then, 95 μl of EIA-positive hybridoma conditioned medium was added to each
And incubated for 45 minutes at RT. TB
S-Tween (20 mM Tris pH 7.5, 50 m
M NaCl, 0.02% v / v Tween 20)
Wash the wells three times using 250 μL per wash,
Furthermore, TBS-Tween (20 mM Tris pH7.
5, 0.5 M NaCl, 0.09% v / v Tween
n 20) using 250 μL per wash
Wash twice and aspirate through the membrane after each addition
Was. Goat anti-mouse IgG H and L chain specific HRP binding
Second antibody (diluted 1: 1000 in TBS-Tween)
Tadashi: Boehringer Mannheim Bioc
chemicals, Indianapolis, IN)
Incubate 100 μL for 45 min at RT in each well.
I was vate. Wash the membrane as described above and remove
Enhanced Chemilu prepared
minescent Reagent (ECL reagent; A
mersham Life Sciences, Cor
position, Arlington Height
s, IL) and X-OMAT AR film
(Kodak Scientific Imagin
g, Rochester, New York)
Glowed. After 15 seconds, remove the film from the film cassette.
And developed. Dot of individual hybridoma supernatants
The rating of each hole based on the strength of is from 3+ to 0
Was.

[0047]Example 4 Anti-EPOR antibody binding to EPOR A. Antibody binding to EPOR by BIAcore analysis
body  Surface Plasmon Resonance (SPR) (Fiagerstam
J. et al. Mol. Recognition3, 208
(1990); Malmboro et al., Scand. J.
Immunol.35, 643 (1992))).
Real-time biospecific interaction analysis (real-time
 biospecific interaction
analysis) (BIA, Pharmacia B)
iosensor AB, Uppsala, Sweden
n) is the screen of the ELISA positive monoclonal antibody.
Used for lining.

The soluble HuEPOR prepared as described in Examples 1 and 2 was covalently bonded to the sensor chip CM5 via a primary amine group. HBS (10 mM
HEPES pH 7.4, 150 mM NaCl,
Immobilization was carried out in 3.4 mM EDTA, 0.05% BIAcore surfactant P-20) at a flow rate of 5 μL / min. First, EDC (400 mM N-ethyl-N- (dimethylamine-propyl) carbodiimide in water,
Pharmacia Biosensor AB) and N
HS (100 mM N-hydroxysuccinimide in water, Pharmacia Biosensor AB)
The carboxylated matrix of the sensor chip was activated by injecting 40 μL of a 1: 1 mixture with Soluble EPOR (10 mM sodium acetate pH
65 μL of 50 μg / mL in 4.0) were injected and immobilized on the sensor chip. Extra reactive groups on the sensor chip were replaced with ethanolamine (Pharmacia Bio).
sensor AB) inactivated by injection of 50 μL.

Each analysis cycle involved the injection of 20 μL of hybridoma supernatant followed by the injection of 10 μL of 10 mM HCl for regeneration of the chip. The SPR response is measured in resonance units (Resonance Un
it) (RU). For most proteins, 1
000 RU corresponds to a surface concentration of about 1 ng / mm ² . Table 1 shows the results of screening 96 holes that were positive by EIA. In these experiments, the background is typically about 20 RU. Binding to EPOR is significant above 50 RU.

[0050]

[Table 1]

[0051]

[Table 2]

[0052]

[Table 3]

Tissue culture media conditioned by hybridomas secreting the antibodies shown in the table were tested in the assays shown in the table. The supernatant containing all the antibodies shown in the table showed a positive signal in the ELISA assay. +++, ++, + indicate a positive response, and +++ indicates the response with the greatest effect.
-Represents a response less than or equal to the response of the control medium. NT indicates that the sample was not tested. ?
Indicates a sample that was unable to show a response.

(1) Antibodies 1-61 were used in mice # 7 and # 8
Antibody. Antibodies 62-96 are antibodies from mouse # 9.

(2) Ma using a via core chip to which sHuEPOR is attached
Response unit by b.

(3) The competition on BIACORE was for anti-sHuEPOR Mab 1G2.
After incubating sHuEPOR bound to the sensor chip with 1G2, the effect on Mab binding was quantified as compared to binding to EPOR without prior incubation with 1G2. A is the antibody whose binding is completely (80-100%) blocked.
The antibody whose binding is blocked by 50-80% is C. The antibody whose binding is blocked by less than 50% is B.

(4) Shows the value of the antibody that gave cells a higher average fluorescence than the control (12, 73). "-" Indicates an antibody with a mean fluorescence lower than or equal to the control.

(5) Inhibition of ³ H uptake by UT7-EPO cells. 30 units of EPO and various amounts of antibody were incubated with UT7-EPO cells.
After overnight incubation, cells were pulsed with ³ H thymidine and the amount of incorporated counts quantified. A positive response was defined as a "response with a gradual decrease in uptake as the amount of antibody increased."

(6) Stimulation of ³ H uptake by UT7-EPO cells. Various amounts of antibody were incubated with UT7-EPO cells. After incubating overnight,
Cells were pulsed with ³ H thymidine and the amount of incorporated count was quantified. A positive response was defined as a "response with a gradual increase in uptake as the amount of antibody increased."

(7) Inhibition was confirmed at a concentration higher than the concentration required for activation.

[0061]B. Epitope competition analysis  Hybridizes the sensor chip immobilized with sHuEPOR
Saturate by injecting 265 μL of 1G dormer supernatant
It was possible. 1G2 is the procedure described in the third embodiment.
Generated for sHuEPOR using
Is an internal antibody. Each analysis cycle is 1G26
With or without epitope saturation by injection of 5 μL
Injection of 20 μL of hybridoma supernatant without saturation
Included. "After 1G2 saturation, hybridoma supernatant
Signal (RU) when 20 μL of liquid is injected)
"When only 20 μL of hybridoma supernatant is injected
Of the binding signal (RU) of the
Defined as% cking. Blocking 80-100
% Antibody is designated as "Group A" and blocking 50%
The following antibodies are referred to as "Group B" and have a blocking 50
-80% of antibodies are designated "Group C". Display analysis results
It is shown in FIG.

[0062]C. Fluorescence activated cell sorting (FACS)
D40 on transfected CHO cells by analysis
Antibodies that bind to EPOR  The hybridoma supernatant generated against EPOR was
FACS analysis showed that pDSRαEPORd40
EPO receptor on the surface of transfected CHO cells
Was tested for binding to d40 EPO receptor
Cells transfected with DNA encoding
Was constructed as described in Example 1. CHO / EP
OR cells were scraped from the tissue culture dish and PBS /
Resuspended as a single cell in a solution of 0.5% BSA,
Then, about 3 x 10 cells⁵96 holes round bottom at the rate of individual / hole
Dispensed into plates. 1000xg of this plate
For 5 minutes. PBS / BSA after centrifugation
The supernatant was removed and each of the pelleted cells was
Medium or one of the EPOR hybridoma supernatants
Was resuspended. Incubate cells at 4 ° C for 1 hour
Was. After incubation, cells are washed with PBS / BSA
Wash, then fluorescein isothiocyanate
(FITC) labeled goat anti-mouse monoclonal antibody (S
southern Biotech, Birmingha
m Ala. ). Cells are again incubated at 4 ° C for 1
Incubated for hours, washed and analyzed by FACS.
Of the 96 supernatants tested, 5 were used as control cultures.
Had a greater mean cell fluorescence than ground (see Table 1).
Want). Mab71 shows the highest level of fluorescence and its
Next, Mab 74, Mab 58, Mab 73, M
ab 87 was followed. All other supernatants tested were
It showed no fluorescence above that of the control medium.

[0063]Example 5 Purification of anti-EPOR antibody and Fab fragment A. Ascites production  Balb / c mice (Charles R
vers Laboratories, Wilmin
gton, MA) for 7 to 10 days after cell line infusion
Before, 2,4,10,14-tetramethyl-pentadeca
(Pristane; Sigma, St. Louis)
s, MO). 0 .0 for each mouse.
One 5 mL intraperitoneal injection was performed. Ascites is in each of its cell lines
Individual cell lines to be prepared for each
Was injected into 10 to 20 animals for each cell line.

The hybridoma cell lines grown in complete medium until confluence was obtained were washed once with D-PBS, and then washed with Neubauer Hemacytometer.
Counted using er. Each mouse was intraperitoneally injected with 10 ⁷ cells, and thereafter, was allowed to ingest Rodent Lab Chow and water arbitrarily until ascites occurred, to survive. Mice were observed for maximal ascites formation, killed with CO ₂ , punctured using an 18G needle inserted into the ascites-filled abdominal cavity, and ascites collected.
225 xg in a microcentrifuge (Eppendorf)
Clarified by centrifugation at for 15 minutes or 3 minutes. Thereafter, store the 4 mL aliquots at -20 <0> C,
Then, it refine | purified by the protein A column chromatography.

[0065]B. Monoclonal antibody protein A
Purification  From 4 mL of ascites fluid or 10 mL of hybridoma conditioned medium
Immunoglobulin is analyzed by protein A column chromatography
And purified. Bio-Rad Monoclona
l Antibody Purification S
system II (MAPS II; Bio-Rad
Laboratories, Richmond, CA)
It was used. Briefly, Affi-gel P
Dissolve 5 mL of the protein-A suspension in 1 × 10 cm
And loaded into a glass column. Protein A gel
Load with approximately 30 mL of D-PBS, then bind
Immersion liquid (MAPS II Binding Buffer)
r; Bio-Rad) through a column
did. Afterwards, ascites or 1: 1 diluted with binding buffer
Add conditioned medium to the top of the column and allow
I made it. Immunoglobulin conjugated to protein A
Later, the unbound fraction was removed. Then,
Wash ram with 30 mL binding buffer to remove unbound protein
From the column and absorb at 280 nm less than 0.01.
Brightness was obtained. Then the immunoglobulin-containing flux
To Bio-Rad Elution Buffer
Eluted in about 30 mL. This fraction is referred to as DP
Dialysis against 4 L of BS overnight at 4 ° C.
Buffer exchange was performed. The resulting PBS equilibrated immunological group
Roblin, Centricon Concentra
tor Unit (Amicon Inc., Beve)
(ly, MA) by centrifugation at 1700 xg.
And concentrated.

[0066]C. Fractionation of antibody binding domain  Pierce ImmunoPure Fab Pre
parent Kit (Pierce Chemi
cal Company, Rockford, IL)
Using immunoglobulins purified with protein A,
The two component parts, the crystallizable fraction
(Fc) and antibody-bound fraction (Fab).
Painted. 20 mM purified protein A immunoglobulin
Acid / 10 mM EDTA buffer (pH 7.0)
It was dialyzed and then concentrated to about 20 mg / mL. 10
mg of immunoglobulin was fractionated. Immobilized papa gel
System in 12 mL of phosphate buffer as supplied.
Washed twice with digestion buffer containing 42 mg of in. afterwards
Add the immunoglobulin sample to the gel and shake
Incubate overnight at 37 ° C. on the instrument. protein
A purification allows the solubilized Fab to be combined with Fc and undigested immunoglobulin.
Separated from Roblin. Unbound fraction
Time points were collected as Fab samples. This non-bonded part is
Dialyzed against 4 liters of D-PBS at 0 ° C overnight.
Concentrated as described.

[0067]Example 6 Mapping of Mab 71 epitope on EPOR  Residues 1 to 224 of the human EPO receptor (where the residues
1 is proline and residue 224 is aspartic acid
), An overlapping synthetic peptide of 17-30 amino acids in length
A peptide was prepared. 10 different peptides, both ends
6 amino acids overlapped. Of these peptides
Sequences and their respective peptides in the human EPO-R amino acid sequence
The position of the tide is as follows.

SE-1 PPPNLPDPKFESKAALLAARGPEELCFTE (residues 1-30) SE-2A LLCFTERLEDLVCFWEEA (residues 25-42) SE-2B CFWEEAASAGVGPGNYSF (residues 37-54) SE-3 PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (residues 49-78) SE-4 TARGAVRFWCSLPTADTSSFVPLELRVTAA (residues 73-102) SE-5 LRVTAASGAPRYHRVIHINEVVLLDAPVGL (residues 97-126) SE-6 DAPVGLVARLADESGHVVLRVLPPPETPMT (residues 121-150) SE-7 PETPMTSHIRYEVDVSAGNGAGSVQRVETFTFA RG-RV TFERTF-RSV TF-RSV 198) SE-9 FAVRARMEAPSFGGFWSAWSEPVSLLTPSDLD (residues 193-224) Polystyrene well plate (Costar, Cambridge)
dge, MA) in a carbonate-bicarbonate buffer (0.0
15M Na ₂ CO ₃ , 0.035M NaHCO ₃
pH 9.2) in a concentration of 100 μg / mL, 20 μg / m
L and 0.8 μg / mL of the EPO-R peptide. The plate was incubated at room temperature (RT) for 2 hours, then at 4 ° C. overnight. Under the same conditions, soluble HuEPOR was added to a concentration of 10 μM.
g / mL, 2 μg / mL, 0.4 μg / mL, and
Coated as a positive control at 0.08 μg / mL. Plate at RT with 5% BSA in PBS
After blocking for 0 min, 5 μg /
Purified Ma as described in Example 5 at a concentration of mL
The plate was incubated with b71 for 2 hours at RT. Wash buffer (Kirkegard an
d Perry Labs, Inc. ) And then wash the plate with horseradish peroxidase (B
RT together with a 1: 1000 dilution of goat anti-mouse IgG conjugated with Ehringer Mannheim
For 1 hour. Wash the plate, AB
TS substrate solution (Kirkegard and Perr
y Labs, Inc. ). Colorimetric analysis was performed at 405 nm. Mab binding to the above synthetic peptide
The results are shown in Figure 1 and indicate that Mab 71 binds a significant amount of peptide SE-3 (amino acid residues 49-78 of human EPO-R) compared to when it binds to the other peptides tested. ). This indicates that for regions of human EPO-R that include residues 49-78 or that overlap residues 49-78, the Mab
71 indicates binding.

[0069]Example 7 Activity of anti-EPOR antibody in cell proliferation assay  Antibodies in the conditioned medium prepared as described above were purified using UT7-E
By PO cells (Komatsu et al., Supra)³H-thin
It was assayed for its ability to stimulate gin uptake.
UT7-EPO cells respond to EPO on their cell surface
To express the human EPO receptor. UT7-EPO cells
Using a growth medium (L-glutamine, 25 mM HEPES)
Buffer and 3024 mg / L sodium hydrogencarbonate
And alpha-thioglycerol or beta
-1x Iscov without mercaptoethanol
e modified Dulbecco medium (GIBCO) / 10%
v / v fetal calf serum / 1% v / v L-glutamine-peni
Syrin-streptomycin solution (Irvine Sc
inentific) / 1 unit / mL rHuEPO)
And about 3 × 10⁵Cells were grown to cells / mL. Centrifuge cells
(Approximately 500 xg) and with phosphate buffered saline
Wash twice, assay medium (1 × without L-glutamine)
 RPMI medium 1640 (Gibco) / 1% L-G
5 x 10 in glutamine / 4% fetal calf serum⁴Cells / m
L. More than 5-fold dilution in assay medium
100 μL of test sample or EPO standard (rHuEPO)
Added to the wells of a 96-well microtiter plate. afterwards
Add 50 μL of cells (5000 cells / well)
5% CO at 37 ° C in a humidified incubator₂To
And incubated. After 72 hours, assay medium
Methyl diluted 1: 100 in the ground³H-thymidine
(1 mCi / mL; 20 Ci / mmol)
I got it. Cells are further incubated at 37 ° C. with 5% CO 2₂4 hours in
Incubated. The labeled cells are harvested from the PHD cell harvest.
Star (Cambridge Technology)
Inc. ) And gas using deionized water (as wash water)
Collected on lath fiber filter mats. Filter
Final washing with 2-propanol, followed by dehydration,
Beckman Model LS6000IC Cinch
It was counted with a ration counter.

Conditioned media from tissue culture plates containing the anti-EPOR Mab was tested as described above for its ability to stimulate growth. Samples were tested at several concentrations. Positive responses were identified as "thymidine incorporation was determined to be at background level 2
Stimulation more than 2-fold, and when the sample is diluted, there is also a decrease in stimulation ". As shown in Table 1, two of the 24 samples tested showed a positive response (Mab 71, Mab 73). The four samples may have weak stimulatory activity ("?" In Table 1). The remaining samples did not result in a large increase in thymidine incorporation above background. Polyclonal sera from mice used to generate monoclonal antibodies also stimulated thymidine incorporation. This means
This suggests that the polyclonal antibody in the serum could also stimulate the proliferation of UT7-EPO cells.

The supernatant was also tested for its ability to inhibit EPO-induced stimulation of thymidine incorporation by UT7-EPO cells. Cells were incubated with 25 units / mL rHuEPO and various amounts of antibody containing conditioned media. Thymidine incorporation was measured as described above.
Table 1 shows the results. Most antibodies did not differ significantly from the control medium. Among the antibodies that showed inhibition of thymidine incorporation, two samples (Mab 58 and Mab 73) showed a clear inhibition, while 3
Samples (Mab 65, Mab 88, Mab 8
9) showed possible inhibition. Mab 73 showed inhibition at the highest dose, but lower doses stimulated thymidine incorporation above control values.

[0072]Example 8 EPOR activity by anti-EPOR antibody and fragment
Conversion A. UT7-EPO proliferation assay  The Mab 71 and the Mab 73 are communicated as described in the fifth embodiment.
And purified. Proliferation activity was measured using UT7 as described in Example 7.
-Quantified in EPO thymidine incorporation assay. Mab
 Both 71 and Mab 73 are rHuEPO
Similarly, in a dose-dependent manner, UT7-EPO
Gin uptake was stimulated (see Example 2).
Higher doses of Mab 71 reduced activity. Stimulating activity
The peak is 1-2 μg / mL for Mab 71.
In dose, from 100 μg / mL for Mab 73
Observed at higher doses. Non-neutralizing control antibody (anti-EPO M
ab F12) did not stimulate thymidine incorporation
However, this means that the stimulation of thymidine incorporation
This indicates that the antibody is specific to a body antibody.

[0073]B. EPO low temperature displacement assay  Antibodies to the EPO receptor include a region to which EPO binds
Can bind to the same region. To test this possibility
Next, a cold displacement assay was performed using OCIM1 cells.
Was. OCIM1 cells are cells of human origin,
It is known to contain EPO receptors on the surface (Br
audy et al., Proc. Nat. Acad. Sci. U
SA85, 6517 (1988)). The cells are
M1 cell culture medium (Iscover's modified Dulbecco culture medium)
Ground (IMDM) / 10% fetal calf serum / 1% pen-
strep-fungizone), about 2-5 × 1
0⁵Cells were grown to cells / mL. Collect cells by centrifugation
And binding buffer (RPMI 1640/1% BSA / 2
Wash twice in 5 mM HEPES pH 7.3),
Followed by 0.1% azide and 10 μg / mL cytosolic
1-2 x 10 in binding buffer containing Syn B⁷Cells / mL
Was resuspended. Then, in a 96-well tissue culture plate
Of cells (100 μL) and 10 μL of sample¹²⁵IE
PO (Amersham high specific activity; 3000 Ci / mm
ol, 2μCi / mL) together with a humidified tissue culture incubator.
Incubated at 37 ° C in beta. 3 hours later
First, the cells were placed in a phthalate oil (60:
40 (v / v) dibutyl / dinonyl phthalate)
And centrifuged. Place the tube containing the cells on dry ice-
Quickly freeze in ethanol bath and collect cell pellet
After doing, LKB 1277 gammamaster
Counting was performed with an automatic gamma counter.

FIG. 3 shows the results of a low-temperature substitution experiment. As the amount of unlabeled rHuEPO added increased, the amount of ¹²⁵ I-EPO displaced from the EPO receptor increased. Similarly, Mab 71 purified as described in Example 5
^{The amount of 125} I-EPO substituted increased with increasing amount of antibody. In this case, the same amount of ¹²⁵ I-EP
Mab 71 required about 4,000 times the amount of rHuEPO to replace O. In contrast, Ma
b 73 showed signs of displacement at the highest dose,
The non-neutralizing anti-rHuEPO Mab (F12) showed no significant substitution. These results indicate that Mab F12
It does not prevent the binding of EPO to the EPO receptor, but Ma
b 71 and Mab 73 have been shown to prevent this binding. This result further indicates that Mab 71
It has been shown to bind to the PO receptor and activate it at or near the EPO binding site.

[0075]C. Activity of Mab 71 and Fab 71
comparison  The EPO receptor fragment of Mab 71 was prepared in Example 5
Prepared as described in. As shown in FIG.
Gel electrophoresis (Laemmmli et al., Nature
 227, 680 (1970)).
It has become. 0.7 M 2 in sample buffer containing 2% SDS
-Boil the sample with or without mercaptoethanol
Raise, non-reducing (no 2-mercaptoethanol) tamper
Protein and reduced (2-mercaptoethanol) protein
Prepared individually, 12.5% acrylamide SDS gel
Electrophoresed. Stain the gel with Coomassie Blue
And visualized the protein. Mobility of protein standards
By comparing the mobility of the protein against
And estimated the size of the protein. Mab 71 and M
When ab 73 was run under reducing conditions, the light chain
Separated into chains. The heavy chain was approximately 52kDA. Mab
 The light chain in the case of 73 is Mab 71 (28.5 kD
It was slightly smaller than in case a) (28 kDa). F
The ab fragment also had two chains. That is,
In the case of Fab 71, 28.3 kDa and 27.3 k
Da, and in the case of Fab 73, 27.5 kDa
And 26.5 kDa. These Fab fragments
Was run under non-reducing conditions, the Fab 71
The size is about 48 kDa, and the size of Fab 73 is about
It was 47 kDa. This means that the Fab fragment
Monovalent, the complex has one L chain and one H chain
It is shown that. In contrast, non-reducing SDS
The mobility of Mab 71 and Mab 73 on the
Their size was shown to be about 200 kDa. this
This means that these Mabs are bivalent and the heavy and light chains
It indicates that there is one by one.

The monovalent Fab 71 fragment is EPO
To determine whether to activate the receptor, Mab
The 71 and Fab 71 fragments were incubated with UT7-EPO cells and thymidine incorporation was determined as in Example 7.
The measurement was performed as described in the above. As shown in FIG. 5, rHu
Both EPO and Mab 71 stimulated thymidine incorporation. However, the monovalent Fab 71 fragment did not stimulate thymidine incorporation. An unrelated receptor (He
The control monoclonal antibody against r2 / neu) also did not stimulate thymidine incorporation. This indicates that the antibody must be bivalent to activate the receptor.

[0077]D. Mab in the presence of rHuEPO
Of Thymidine Uptake by Fab 71 and Fab 71  Mab 71 shows that EPO binds to the EPO receptor.
The fact that they inhibit Mab 7 in the presence of EPO
1 may not activate the EPO receptor
Incited. To confirm this possibility, 30 units
s / mL rHuEPO with varying amounts of purified Mab 7
1, Fab 71 or Mab control (Her2 / ne
UT7-EPO cells together with an antibody against
Was added. Thymidine incorporation was measured as described above.
Was. As shown in FIG. 6, both Mab 71 and Fab 71
The higher dose inhibited thymidine incorporation. But about
At doses between 30 μg / mL and about 3000 μg / mL
Shows that Mab 71 is stimulated only by rHuEPO
Thymidine to a level above the level of thymidine incorporation
Stimulated uptake. Fab 71 and control antibody were
Did not show any action. This means that Mab 71 and r
HuEPO has additional effect on EPO receptor activation
Has been shown.

[0078]Example 9 Stimulation of erythroid colony formation by anti-EPOR antibody  Purified Ma in the formation of red blood cells from precursors in peripheral blood
To determine if b71 stimulates, BFUe
The assay was performed. To purify erythroid progenitor cells,
Normal human donors are ligated according to standard protocols.
Lymphopherase was used. Rin
Hanfer's cells (250 mL) were transferred to Hank's
Balanced Salt Solution (HB
SS) Washed with 250 mL. Resuspend cells in HBSS
Turbidize, 50 gradient on Ficoll-paque
Separated by density centrifugation at 0xg for 30 minutes. Low
Density cells (LD) are collected from the gradient and HBSS 500
Washed with 0.5 mL bovine serum albumin and 5 mM
5 × 10 5 in PBS supplemented with EDTA⁸Cells / m
L was resuspended. After that, Miltenyi
 CD34 manufactured by Biotech GmbH
Progenitor Cell Isolation Kit (QBe
nd / 10) was used to purify LD cells. Short time
Of cells labeled with anti-CD34 monoclonal antibody
And then, according to the protocol, transfer the cells to magnetic microspheres.
Was bound. Next, the pre-filled MiniMac
Pass labeled cells through the separation column
Is washed and CD34⁺Cells were eluted from the column. This
Is repeated to obtain a higher purity CD34.⁺Cells
Obtained. Iscover et al. (J. Cell. Physiol
 83, 309 (1974)).
An in vitro assay was performed with all modifications. culture
The medium was transferred to Gibco BRL (human bone marrow stem cell proliferation kit).
G; Grand Island, NY). 35x
Duplicate 1 mL of sample on 100 mm tissue culture plate
To rate, add the excess 3 mL to 17 x 100
Prepared in sterile polystyrene tubes. Each chew
2.5 mL of stem cell growth medium, CD34⁺Cells (9
0.1 mL, resuspended at 000 cells / mL, stem cell factor
0.015 mL (20 μg / mL), sample and stem
Add 0.385 mL equivalent of cell dilution medium combination
Was. Shake the tube vigorously, settle and foam
Left it. After that, with 17 × 1-1 / 2 needle
The contents were aliquoted using a 3 mL syringe. Humidification
Plate at 37 ° C. in a tissue culture incubator for 10
% CO₂Incubated below. Red blood cell colony
(Range color to red) was scored after 21 days. EPO or
Plates lacking Mab 71 contain erythroid colonies
Was not seen. rHuEPO (30 units /
Plate) is 400 colonies per plate
Excess. Mab 71 also has red blood cell colonies
Spawned. Peak activity was observed at 2-6 μg / mL
Was. This result indicates that Mab 71 forms erythroid colonies.
Is shown to stimulate.

The activity of purified Mab 71 was also tested for its ability to form erythroid colonies using serum-free growth conditions in methylcellulose. CD34 ⁺ cells were isolated as described above and serum-free growth described in co-pending and commonly owned US patent application Ser. No. 08 / 079,719, which is incorporated herein by reference. The medium was used for incubation with the following changes. Assay tubes were prepared without using extracellular matrix molecules, hydrocortisone, and growth factors EGF, FGF, and PDGF. As described above, a 3 mL sample was prepared for plating duplicate 1 mL samples on the plate. 100x stock solution (2
-Mercaptoethanol, nucleosides, cholesterol, sodium pyruvate, Hu-transferrin,
Lipid, Hu-insulin) 0.030 mL each, deionized BSA (15%) 0.4 mL, SCF (20 μg / m
L) 0.015 mL, CD34 ⁺ cells (300,000
Cell / mL resuspension) 0.1 mL, methylcellulose (2.3%) 1.080 mL, and a combination of IMDM and a sample equivalent to 1.195 mL.
L) was placed in each of the tubes. Thereafter, the plates were incubated as described above and
After a day, the colonies were scored. Red blood cell colonies were observed when grown in the presence of EPO or Mab 71, but no red blood cell colonies were found when these two factors were not present. One example of a red blood cell colony type is shown in FIG. Colonies incubated with 25 units of rHuEPO had a similar appearance to colonies grown with 2.1 μg / mL purified Mab 71. The higher the dose of rHuEPO, the larger the colonies. The dose response curve is shown in FIG. Ma
b71 showed peak activity at doses ranging from 1 μg / mL to 5 μg / mL. At lower or higher doses, the number of red blood cell colonies was reduced. Her
The control monoclonal antibody to 2 / Neu did not give rise to any colonies in this dose range. This result indicates that Mab 71 will stimulate the formation of erythroid colonies from erythroid progenitors and that no additional serum is required. Therefore, Mab
71 is capable of stimulating the differentiation of red blood cell precursors into red blood cells.

Although the present invention has been described with reference to preferred embodiments, it is to be understood that variations and modifications can be made by those skilled in the art for such embodiments. It is therefore intended that the appended claims cover all equivalent variations that fall within the scope of the invention as claimed.

[0081]

[Sequence List] SEQ ID NO: 1 Sequence length: 42 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA sequence CTCCAAGCTT GCCGTCACCA TGGACCACCT CGGGGCGTCC CT 42 Sequence number Sequence length: 35 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA sequence CAGGTCTAGA TTACTAGGGA TCCAGGTCGC TAGGC 35 Sequence number Sequence characteristics Sequence length: 27 Sequence type: Nucleic acid Number of strands: One Main chain Topology: linear Sequence type: cDNA sequence AGGTCGACTA CTAGTAGTCA GTTGAGA 27

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Mab against the indicated concentrations of synthetic peptides.
7 shows the results of an ELISA assay measuring the binding of 71. The peptides correspond to the amino acid residues of the human EPO receptor shown in the figure. Residue 1 is an amino-terminal proline found in the EPOR secreted upon cleavage of the leader sequence.

FIG. 2: Different amounts of rHuEPO protein and purified Ma on ³ H thymidine incorporation by UT7-EPO cells.
b shows the effect of 71 and 73.

FIG. 3. Varying amounts of r for inhibition of ¹²⁵ I EPO binding to EPO receptor on the surface of OCIM1 cells.
HuEPO protein, Mab 71, Mab 73,
Alternatively, a non-neutralizing control Mab against EPO (Mab F1
The effect of 2) is shown.

FIG. 4. Coomassie-stained SD of monoclonal antibody Mab 71 and 73 purified samples and monoclonal antibody fragment (Fab) purified samples derived from these antibodies.
2 shows an S gel. The sample was run under reducing (to which 2-mercaptoethanol was added) or non-reducing (to remove 2-mercaptoethanol) conditions.

FIG. 5. Different amounts of purified rHuEPO protein, Ma, on ³ H thymidine incorporation in UT7-EPO cells.
The effect of b71 or Fab 71 is shown.

FIG. 6. Recombinant human EPO (rHuEPO) 30 mun
4 shows the effect of varying amounts of purified Mab 71 or Fab 71 on ³ H-thymidine incorporation of UT7-EPO cells, also added in ITS / ml.

FIG. 7. EPO or Mab under serum-free growth conditions
It was grown in methylcellulose for 21 days in the presence of 71 shows a photograph of purified CD34 ⁺ + cells from peripheral blood. The photo shows 500 units / ml EPO
(A), 25 units / ml EPO (B), or
Cells incubated with 2.1 μg / ml Mab 71 (C).

FIG. 8. Varying amounts of purified rHuEPO protein, M, on erythroid colony formation from erythroid precursors when grown under serum-free growth conditions in soft agar.
5 shows the effect of control monoclonal antibodies on ab 71 and Her2 / neu.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12N 15/02 G01N 33/53 D C12P 21/08 33/566 G01N 33/53 33/577 B 33/566 C12N 5 / 00 B 33/577 15/00 C // (C12P 21/08 C12R 1:91)

Claims (26)

[Claims] 1. An antibody or a fragment thereof that activates an erythropoietin receptor. 2. The antibody according to claim 1, wherein the erythropoietin receptor is a mammalian erythropoietin receptor. 3. The antibody according to claim 1, wherein the erythropoietin receptor is a human erythropoietin receptor. 4. The antibody according to claim 1, which is a monoclonal antibody. 5. The antibody according to claim 1, which is a humanized antibody. 6. The antibody according to claim 1, which is a human antibody. 7. The antibody of claim 1, having a detectable label. 8. A hybridoma cell line capable of producing said monoclonal antibody according to claim 4. 9. The hybridoma cell line ATCC No.
HB11689 or ATCC No. An antibody or a fragment thereof that recognizes an epitope on the erythropoietin receptor, which is recognized by a monoclonal antibody produced by HB11690. 10. The antibody according to claim 9, which activates an erythropoietin receptor. 11. The antibody according to claim 9, wherein the erythropoietin receptor is a human erythropoietin receptor. 12. The antibody according to claim 9, which is a monoclonal antibody. 13. The antibody according to claim 9, which is a humanized antibody. 14. The antibody of claim 9, which has a detectable label. 15. A hybridoma cell line capable of producing the monoclonal antibody according to claim 12. 16. The hybridoma cell line ATCC N
o. HB11689 or ATCC No. HB1169
Antibodies produced by 0. 17. The hybridoma cell line ATCC N
o. HB11689 or ATCC No. HB1169
0. 18. A method for detecting an erythropoietin receptor that can be activated in a biological sample, comprising: (a) contacting the sample with an antibody according to claim 1 or 9; (B) detecting activation of the receptor by the antibody, thereby determining the presence of an activatable erythropoietin receptor. 19. An antibody comprising the antibody according to claim 1 or 9.
A kit for detecting an activatable erythropoietin receptor in a biological sample. 20. A method for modulating the endogenous activity of an erythropoietin receptor in a mammal, comprising administering an effective amount of the antibody according to claim 1 or 9 effective for modulating the activity of the erythropoietin receptor. 21. The method of claim 2, wherein the modulation of erythropoietin receptor activity controls erythroid progenitor cell proliferation or differentiation.
The method according to 0. 22. A method for treating anemia in a patient, comprising administering a therapeutically effective amount of the antibody of claim 1 or 9. 23. A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to claim 1 in a pharmaceutically acceptable adjuvant. 24. The composition according to claim 23, wherein said antibody is a monoclonal antibody. 25. The antibody of claim 2, wherein said antibody is a humanized antibody.
5. The composition according to 4. 26. The antibody of claim 24, wherein said antibody is a human antibody.
A composition according to claim 1.